Calcium stimulated the endogenous phosphorylation of several synaptosomal cytosolic proteins. The effects of calcium were both concentration and time dependent and were most apparent for proteins with molecular weights (Mr) of 50,000, 55,000 and 60,000. Exogenous calcium (1.0-100 micron M) enhanced the net incorporation of phosphate into protein by as much as 23-fold. The antipsychotic, fluphenazine (1.0-100 micron M), caused a concentration dependent decrease in calcium stimulated protein phosphorylation. When the heat-stable calcium binding protein, calmodulin, was removed from synaptosomal cytosol by affinity chromatography on fluphenazine-sepharose, calcium-stimulated protein phosphorylation was abolished. Responsiveness to calcium could be stored by the addition of calmodulin to the phosphorylation assay. These results indicate that calcium dependent protein kinases are of major importance in regulating the phosphorylation of specific cytosolic proteins in neuronal tissue.